Method for identification of modulators of pre-adipocyte differentiation

ABSTRACT

The invention relates to a method for identification of a compound which modulates pre-adipocyte differentiation comprising testing whether the compound modulates the activity and/or amount of O/E-1 and/or O/E-2 and/or O/E-3. The invention also relates to use of a compound able to modulate the activity or amount of O/E-1 and/or O/E-2 and/or O/E-3 in preparation of a medicament for the treatment or prevention of atherosclerosis, dyslipidemia, IRS, and type 2 diabetes.

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application No. PCT/GB02/04479, filed Oct. 3, 2002, whichclaims priority from United Kingdom Patent Application No. 0123962.3,filed Oct. 5, 2001, and United Kingdom Patent Application No. 0214814.6,filed Jun. 27, 2002, the specifications of which are incorporated byreference herein. International Application No. PCT/GB02/04479 waspublished under PCT Article 21(2) in English.

The invention relates to methods of screening test compounds for theirability to modulate pre-adipocyte differentiation by measuring theiractivity as a modulator of O/E-1 and/or O/E-2 and/or O/E-3 activity orexpression. The invention also relates to the use of active modulatorsof O/E-1 and/or O/E-2 and/or O/E-3 activity or expression in stimulationof pre-adipocyte differentiation and hence also in the treatment orprevention of atherosclerosis, or insulin resistance syndrome, ordyslipidemia, or type 2 diabetes. The invention also relates to the useof active modulators of O/E-1 and/or O/E-2 and/or O/E-3 activity orexpression in inhibition of pre-adipocyte differentiation and hence alsoin the treatment or prevention of obesity. The development of celllineages with specialized phenotypes is essential for all multicellularorganisms. Each specific cellular phenotype is determined by the set ofgenes expressed and this can be regulated by both intrinsic andextrinsic factors. The most studied way of controlling gene expressionis through transcriptional regulation. Gain and lack of functionexperiments have also shown that numerous transcription factors arecritical for development of many cell types. For example, the myogenictranscription factors Myf-5, MyoD, myogenin essential for muscle celldevelopment (Massari and Murre (2000) Mol Cell Biol 20, 429-40), thenuclear hormone receptor PPARγ for adipocytes (Rosen et al. (2000) GenesDev. 14, 1293-307) and the E2A transcription factors for B-lymphocytedifferentiation (Liberg and Sigvardsson (1999) Crit Rev Immunol 19,127-53). The molecular mechanisms controlling the development ofadipocytes have been studied extensively through the use of immortalizedmurine preadipocyte cell lines such as 3T3-L1 and F442A and also by gainof function experiments in NIH/3T3 fibroblasts. These preadipocytes canbe induced by hormonal stimulation to differentiate into adipocytes thatresemble cells found in white adipose tissue. Using 3T3-L1 as a modelsystem for adipocyte development it has been possible to dissect thisprocess into distinct stages with coordinated expression of specificgenes. These include genes important for lipid metabolism and adipocytefunction as well as secreted hormones involved in controlling adipocytedifferentiation. All these genes are however expressed at laterdifferentiation stages when the mature adipocyte is already formed.Studies of the promoters of these adipocyte specific genes have led tothe isolation of several adipocyte differentiating transcription factorssuch as PPAR, C/EBP and ADD1 that coordinate the development of thiscell lineage. (Cowherd et al. (1999) Semin Cell Dev Biol 10, 3-10; Rosenet al. (2000) Genes Dev 14, 1293-307).

EBF (Early B-cell Factor) also called Olfactory factor-1 (Olf-1) orO/E-1 is a 64 kD transcription factor originally described to beimportant for control of B-lymphocyte specific genes and fortranscriptional regulation of genes in olfactory receptor neurons(Hagman et al. (1993) Genes and Development 7, 760-773; Lin andGrosschedl (1995) Nature 376, 263-7; Wang et al. (1997) J Neurosci 17,4149-58). O/E-1 belongs to the helix loop helix (HLH) family oftranscription factors, which includes proteins such as MyoD, E2A, Mycand NeuroD that are important for development and determination ofseveral mammalian cell lineages (Massari and Murre (2000) Mol Cell Biol20, 429-40). This HLH motif is important for homo- andhetero-dimerization of HLH factors; O/E-1 is however suggested tointeract with natural DNA binding sites only as a homodimer (Travis etal. (1993) Mol Cell Biol 13, 3392-3400). O/E-1 differs from other HLHtranscription factors in that it besides this dimerization domain alsocontains a unique zinc coordination motif that mediates DNA binding to aATTCCCNNGGGAAT (SEQ ID NO: 21) consensus DNA binding site (Hagman et al.(1995) Embo J 14, 2907-16; Travis et al. (1993) Mol Cell Biol 13,3392-3400). O/E-1 also contains two transactivation domains, one withinthe DNA binding domain and one in C-terminal part of the protein thatboth are important for full transcriptional activity (Hagman et al.(1995) Embo J 14, 2907-16). Many of the characterized target genes forO/E-1 also contains binding sites for E-box binding transcriptionfactors like the E2A proteins and O/E-1 has also been shown to act insynergy with these factors to achieve a high rate of transcription(Sigvardsson et al. (1997) Immunity 7, 25-36). The O/E family has beenextremely conserved during evolution and is present in C. elegans(Prasad et al. (1998) Development 125, 1561-8), Drosophila (Crozatier etal. (1996) Curr Biol 6, 707-18), zebra fish (Bally-Cuif et al. (1998)Mech Dev 77, 85-90) and chicken (Nieminen et al. (2000) Scand J Immunol52, 465-9), indicating that these proteins and their role have beenpreserved through selective pressure. In mammals the O/E family of HLHtranscription factors contains three highly conserved members:Olf-1/EBF, Olf-1/EBF-like 2 and 3 (O/E-1, -2 and -3) (Wang et al. (1997)J Neurosci 17, 4149-58). In the mouse O/E-1 is expressed inB-lymphocytes, olfactory neurons, cerebellum and adipose tissue, whileO/E-2 and -3 are more restricted to olfactory neurons (Hagman et al.(1993) Genes and Development 7, 760-773; Wang et al. (1997) J Neurosci17, 4149-58). O/E-1 is the best characterized family member and its rolein the development of B-lymphocytes has been studied extensively. Bypromoter analysis several B-lymphocyte specific target genes have beenidentified that all are involved in forming the pre-B cell receptor andsignaling through this complex. Mice made devoid of O/E-1 by homologousrecombination do also show a complete lack of B-lymphocytes, which showsthat this protein is indeed of great importance for B-lymphocytedevelopment. These mice had however no gross defects in the centralnervous system, indicating a redundancy between the O/E proteins in thebrain (Lin and Grosschedl (1995) Nature 376, 263-7). All experimentsperformed so far indicate that O/E-1 seems to play similar roles inB-lymphocytes in humans and mice (Gisler et al. (2000) Blood 96,1457-64). Roaz is a recently described protein that can interact withO/Es and other transcription factors and function as a negativeregulator of transcription suggesting that there are systems availableto fine tune the activity of the O/E proteins (Tsai and Reed (1997) JNeurosci 17, 4159-69); Tsai and Reed (1998) Mol Cell Biol 18, 6447-56).

The present invention is based on the discovery that expression of O/E-1and/or O/E-2 and/or O/E-3 stimulates adipocyte differentiation anddevelopment.

The invention relates to the use of a variety of procedures for usingO/E-1 and/or O/E-2 and/or O/E-3 in the discovery of modulators of O/E-1and/or O/E-2 and/or O/E-3 function or expression in modulatingpre-adipocyte differentiation and therefore used to ameliorate thedyslipidemia associated with conditions like, but not limited to, theinsulin resistance syndrome including type 2 diabetes and obesity andthus ultimately to prevent cardiovascular morbidity and mortality causedby atherosclerosis.

The invention further relates to pharmaceutical compositions containingsuch a modulator discovered by the methods described in this applicationand the use of the modulator or pharmaceutical composition comprisingsuch modulator in modulating pre-adipocyte differentiation and thereforeused to modify or ameliorate or prevent atherosclerosis or insulinresistance syndrome or dyslipidemia or type 2 diabetes or obesity.

Expression of O/E-1 in NIH/3T3 fibroblasts, 3T3-L1 preadipocytes, ormouse embryonic fibroblasts (MEF) enhances the differentiation of thesecells to adipocytes. Expression of O/E-2 and/or O/E-3 in 3T3-L1preadipocytes enhances the differentiation of these cells to adipocytes.This demonstrates that O/E-1 and/or O/E-2 and/or O/E-3 represents animportant regulatory factor in the adipocyte differentiation.

Adipate differentiation is accompanied by the induction of several genesthat are involved in lipid metabolism such as the adipocyte fatty acidbinding protein (aP2), phosphoenolpyruvate carboxykinase (PEPCK), acylCoA synthetase (ACS), the fatty acid transporter (FATP-1) andlipoprotein lipase (LPL) as well as genes involved in glucoseutilisation such as the insulin dependent glucose transporter GLUT4.Compounds that stimulates the adipocyte differentiating effects of O/E-1may therefore be useful in the treatment of disease conditionscharacterised by increased plasma levels of triglycerides, cholesterol,free fatty acids, insulin, glucose and decreased insulin sensitivity.Examples of such diseases are the insulin resistance syndrome includingtype 2 diabetes and obesity. Lowering of the plasma levels of glucose,triglycerides, cholesterol, free fatty acids may also be beneficial forthe prevention and treatment of atherosclerosis.

It is concluded that treatment with modulators of O/E-1 and/or O/E-2and/or O/E-3 activity or amount can lead to stimulation of pre-adipocytedifferentiation and therefore constitutes a novel treatment fordyslipidemia and insulin resistance syndrome and type 2 diabetes.

Compounds that inhibit the adipocyte differentiating effects of O/E-1and/or O/E-2 and/or O/E-3 may be useful in the treatment of diseasescaused by increased fat accumulation or lipid storage. Examples of suchdiseases are obesity, osteoporosis, and acne.

This invention provides methods for stimulation or inhibition ofpre-adipocyte differentiation comprising the administration of aneffective amount of a compound that modulates the activity or amount ofO/E-1 and/or O/E-2 and/or O/E-3. Modulation of the amount of O/E-1and/or O/E-2 and/or O/E-3 by a compound may be brought about for examplethrough altered gene expression level or message stability. Modulationof the activity of O/E-1 by a compound may be brought about for examplethrough compound binding to O/E-1, O/E-1 homodimers or O/E-1-DNAcomplexes.

In a further aspect of the present invention we provide a method for theprovision of an adipocyte differentiation agent, which method comprisesusing one or several putative modulators of O/E-1 and/or O/E-2 and/orO/E-3 amount or activity as test compounds in one or several proceduresto measure the ability of the test compound to modulate O/E-1 and/orO/E-2 and/or O/E-3, and selecting active compounds for use as agentsable to stimulate and inhibit pre-adipocyte differentiation,respectively.

Convenient test procedures include the use of animal models to test therole of the test compound. These will typically involve theadministration of compounds by intra peritoneal injection, subcutaneousinjection, intravenous injection, oral gavage or direct injection viacanullae into the blood stream of experimental animals. The effects oninsulin sensitivity, lipid profiles, food intake, body temperature,metabolic rate, behavioural activities, body weight and body compositionchanges may all be measured using standard procedures.

Suitable modulators may be identified by initial screening formodulators of O/E-1 and/or O/E-2 and/or O/E-3 activity or amount againstO/E-1 and/or O/E-2 and/or O/E-3 expressing cells or the isolated O/E-1and/or O/E-2 and/or O/E-3 protein or fragment or chimaeirc form thereof.

Preferably the screening is performed using an assay selected from:

-   i) measurement of O/E-1 activity using a reporter gene assay    comprising a cell line which expresses O/E-1 and a reporter gene    coupled to an O/E-1 response element and assaying for expression of    the reporter gene.-   ii) measurement of O/E-1 activity using purified O/E-1 protein or a    fragment thereof, and assaying the interaction between O/E-1 and a    DNA fragment, preferably in an electrophoresis mobility shift assay    (EMSA).-   iii) measurement of O/E-1 activity using purified O/E-1 protein or a    fragment thereof and a dimerisation partner or a fragment thereof,    and assaying the dimerisation of O/E-1, preferably by time resolved    fluorescence resonance energy transfer or by scintillation proximity    assay-   iv) measurement of O/E-1 transcription or translation in a cell line    expressing O/E-1.-   v) measurement of direct compound binding or competitive binding to    O/E-1, preferably by time resolved fluorescence resonance energy    transfer or scintillation proximity assay.

Preferably the screening is performed using an assay selected from:

-   i) measurement of O/E-2 activity using a reporter gene assay    comprising a cell line which expresses O/E-2 and a reporter gene    coupled to an O/E-2 response element and assaying for expression of    the reporter gene.-   ii) measurement of O/E-2 transcription or translation in a cell line    expressing O/E-2.

Preferably the screening is performed using an assay selected from:

-   i) measurement of O/E-3 activity using a reporter gene assay    comprising a cell line which expresses O/E-3 and a reporter gene    coupled to an O/E-3 response element and assaying for expression of    the reporter gene.-   ii) measurement of O/E-3 transcription or translation in a cell line    expressing O/E-3.

Preferably the cell line is a 3T3-L1 preadipocyte cell, a 3T3-L1adipocyte cell, a NIH/3T3 fibroblast, or an embryonic fibroblast.

DNA encoding mammalian O/E-1 and/or O/e-2 and/or O/E-3 may beconveniently isolated from commercially available RNA, brain cDNAlibraries, genomic DNA, or genomic DNA libraries using conventionalmolecular biology techniques such as library screening and/or PolymeraseChain Reaction (PCR). These techniques are extensively detailed inMolecular Cloning—A Laboratory Manual, 2^(nd) edition, Sambrook, Fritsch& Maniatis, Cold Spring Harbor Press.

The resulting cDNAs encoding mammalian O/E-1s and/or O/e-2s and/orO/E-3s are then cloned into commercially available mammalian expressionvectors such as the pcDNA3 series (InVitrogen Ltd etc. see below). Analternative mammalian expression vector is disclosed by Davies et al., Jof Pharmacol & Toxicol. Methods, 33, 153-158. Standard transfectiontechnologies are used to introduce these DNA's into commonly availablecultured, mammalian cell lines such as CHO, HEK293, HeLa and clonalderivatives expressing the recombinant O/E-1 and/or O/e-2 and/or O/E-3are isolated. An alternative expression system is the MEL cellexpression system claimed in our UK patent no. 2251622.

In addition, these cDNAs may be transfected into derivatives of thesecells lines that have previously been transfected with a “reporter”gene. Examples of suitable reporter genes are, but not limited to,esterases, phosphatases, proteases, fluorescent proteins, such as GFP,YFP, BFP, and CFP, luciferase, cloramphenicol acetyl transferase,β-galactosidase, β-glucuronidase. The reporter gene is constructed so asto contain promoter elements that will respond to the activity andamount of O/E-1 and/or O/e-2 and/or O/E-3 in the cell by increasedexpression of the reporter gene which can be measured by the level ofthe reporter gene product expressed.

Preferably the protein is human recombinant O/E-1 or mouse recombinantO/E-1. Preferably the protein is mouse recombinant O/E-2 and mouserecombinant O/E-3. The cDNA and amino acid sequence of human and mouseO/E-1 can e.g. be obtained from the EMBL database accession no. AF208502(human O/E-1) and accession no. L12147 (MMEARLYB) (mouse O/E-1).

These assays may be used to identify low molecular weight compounds thatincrease the activity or amount of O/E-1 and/or O/e-2 and/or O/E-3;these are defined as “activators”.

In addition or alternatively, the same assays can be used to identifylow molecular weight compounds that decrease the activity or amount ofO/E-1 and/or O/e-2 and/or O/E-3; these are defined as “inhibitors”.

The test compound may be a polypeptide of equal to or greater than, 2amino acids such as up to 6 amino acids, up to 10 or 12 amino acids, upto 20 amino acids or greater than 20 amino acids such as up to 50 aminoacids. For drug screening purposes, preferred compounds are chemicalcompounds of low molecular weight and potential therapeutic agents. Theyare for example of less than about 1000 Daltons, such as less than 800,600 or 400 Daltons in weight. If desired the test compound may be amember of a chemical library. This may comprise any convenient number ofindividual members, for example tens to hundreds to thousands tomillions etc., of suitable compounds, for example peptides, peptoids andother oligomeric compounds (cyclic or linear), and template-basedsmaller molecules, for example benzodiazepines, hydantoins, biaryls,carbocyclic and polycyclic compounds (eg. naphthalenes, phenothiazines,acridines, steroids etc.), carbohydrate and amino acids derivatives,dihydropyridines, benzhydryls and heterocycles (eg. triazines, indoles,thiazolidines etc.). The numbers quoted and the types of compoundslisted are illustrative, but not limiting. Preferred chemical librariescomprise chemical compounds of low molecular weight and potentialtherapeutic agents.

In a further aspect of the invention we provide the use of a modulatorof O/E-1 and/or O/e-2 and/or O/E-3 activity or amount as an agent ableto stimulate pre-adipocyte differentiation and thereby modify or orprevent atherosclerosis by means of ameliorating the dyslipidemiaassociated with the insulin resistance syndrome including type 2diabetes and obesity.

In a further aspect of the present invention we provide a method oftreating or preventing atherosclerosis, insulin resistance syndrome,dyslipidemia or type 2 diabetes which method comprises administering toa patient suffering from such a disease a pharmaceutically effectiveamount of an agent, preferably identified using one or more of themethods of this invention, able to stimulate pre-adipocytedifferentiation by modulating O/E-1 and/or O/e-2 and/or O/E-3 activityor amount and thereby modify or ameliorate or prevent theatherosclerosis, insulin resistance syndrome, dyslipidemia or type 2diabetes disease.

This invention further provides use of a compound able to modulate theactivity or amount of O/E-1 and/or O/e-2 and/or O/E-3 in preparation ofa medicament for the treatment or prevention of atherosclerosis,dyslipidemia, IRS, and type 2 diabetes. Preferably the compound is anO/E-1 and/or O/e-2 and/or O/E-3 activator.

In a further aspect of the invention we provide the use of a modulatorof O/E-1 and/or O/e-2 and/or O/E-3 activity or amount as an agent ableto inhibit pre-adipocyte differentiation and thereby modify orameliorate or prevent obesity.

In a further aspect of the present invention we provide a method oftreating or preventing obesity which method comprises administering to apatient suffering from obesity a pharmaceutically effective amount of anagent, preferably identified using one or more of the methods of thisinvention, able to inhibit pre-adipocyte differentiation by modulatingO/E-1 and/or O/e-2 and/or O/E-3 activity or amount and thereby modify orameliorate or prevent obesity.

This invention further provides use of a compound able to modulate theactivity or amount of O/E-1 and/or O/e-2 and/or O/E-3 in preparation ofa medicament for the treatment or prevention of obesity. Preferably thecompound is an O/E-1 and/or O/e-2 and/or O/E-3 inhibitor.

Modulation of the amount of O/E-1 and/or O/e-2 and/or O/E-3 by acompound may be brought about for example through altered geneexpression level or message stability. Modulation of the activity ofO/E-1 by a compound may be brought about for example through compoundbinding to O/E-1 protein. In one embodiment, modulation of O/E-1comprises a compound able to increase the activity of O/E-1.

According to another aspect of the present invention there is provided amethod of preparing a pharmaceutical composition which comprises:

-   i) identifying a compound as useful for treatment or prevention of    atherosclerosis, dyslipidemia, insulin resistance syndrome, type 2    diabetes or obesity according to a method as described herein; and-   ii) mixing the compound or a pharmaceutically acceptable salt    thereof with a pharmaceutically acceptable excipient or diluent.

The pharmaceutical composition can further comprise a PPARγ agonist,examplified by, but not limited to, thiazolidinediones, such as,rosiglitazone, and pioglitazone.

It will be appreciated that the present invention includes the use oforthologues and homologues of the human O/E-1 and/or O/e-2 and/or O/E-3.By the term “orthologue” we mean the functionally equivalent O/E-1and/or O/e-2 and/or O/E-3 in other species. By the term “homologue” wemean a substantially similar and/or related O/E-1 and/or O/e-2 and/orO/E-3 in the same or a different species.

For either of the above definitions we believe the O/E-1s and/or O/e-2sand/or O/E-3s may have for example at least 30%, such as at least 40%,at least 50%, at least 60%, and in particular at least 70%, such as atleast 80%, for example 85%, or 90% or 95% peptide sequence identity. Itis appreciated that homologous O/E-1s and/or O/e-2 and/or O/E-3 may havesubstantially higher peptide sequence identity over small regionsrepresenting functional domains. We include O/E-1s and/or O/e-2s and/orO/E-3s having greater diversity in their DNA coding sequences thanoutlined for the above amino acid sequences but which give rise toO/E-1s and/or O/e-2s and/or O/E-3s having peptide sequence identityfalling within the above sequence ranges. Convenient versions of theO/E-1 include the published sequences such as EMBL database accessionno. AF208502 (human O/E-1) and accession no. L12147 (MMEARLYB) (mouseO/E-1). The O/E-1 is from any mammalian species, including human,monkey, rat, mouse and dog. Preferably the human O/E-1 is used.

Fragments and partial sequences of the O/E-1 and/or O/e-2 and/or O/E-3may be useful substrates in the assay and analytical methods of theinvention. It will be appreciated that the only limitation on these ispractical, they must comprise the necessary functional elements for usein the relevant assay and/or analytical procedures.

The agent of this invention may be administered in standard manner forthe condition that it is desired to treat, for example by oral, topical,parenteral, buccal, nasal, or rectal administration or by inhalation.For these purposes the compounds of this invention may be formulated bymeans known in the art into the form of, for example, tablets, capsules,aqueous or oily solutions, suspensions, emulsions, creams, ointments,gels, nasal sprays, suppositories, finely divided powders or aerosolsfor inhalation, and for parenteral use (including intravenous,intramuscular or infusion) sterile aqueous or oily solutions orsuspensions or sterile emulsions.

Knowledge of the O/E-1 and/or O/e-2 and/or O/E-3 also provides theability to regulate its expression in vivo by for example the use ofantisense DNA or RNA. Thus, according to a further aspect of theinvention we provide an appetite control agent comprising an antisenseDNA or an antisense RNA which is complementary to all or a part of apolynucleotide sequences shown in sequence nos. 1, 3 and 5. Bycomplementary we mean that the two molecules can hybridise to form adouble stranded molecule through nucleotide base pair interactions tothe exclusion of other molecular interactions.

The antisense DNA or RNA for co-operation with polynucleotide sequencecorresponding to all or a part of a O/E-1 and/or O/e-2 and/or O/E-3 genecan be produced using conventional means, by standard molecular biologyand/or by chemical synthesis. The antisense DNA or RNA can becomplementary to the full length O/E-1 and/or O/e-2 and/or O/E-3 gene ofthe invention or to a fragment thereof. Antisense molecules whichcomprise oligomers in the range from about 12 to about 30 nucleotideswhich are complementary to the regions of the gene which are proximalto, or include, the protein coding region, or a portion thereof, arepreferred embodiments of the invention. If desired, the antisense DNA orantisense RNA may be chemically modified so as to prevent degradation invivo or to facilitate passage through a cell membrane and/or a substancecapable of inactivating mRNA, for example ribozyme, may be linkedthereto and the invention extends to such constructs.

Oligonucleotides which comprise sequences complementary to andhybridizable to the O/E-1 are contemplated for therapeutic use. U.S.Pat. No. 5,639,595, Identification of Novel Drugs and Reagents, issuedJun. 17, 1997, wherein methods of identifying oligonucleotide sequencesthat display in vivo activity are thoroughly described, is hereinincorporated by reference.

Nucleotide sequences that are complementary to the O/E-1 and/or O/e-2and/or O/E-3 encoding nucleic acid sequence can be synthesised forantisense therapy. These antisense molecules may be DNA, stablederivatives of DNA such as phosphorothioates or methyiphosphonates, RNA,stable derivatives of RNA such as 2′-O-alkylRNA, or otheroligonucleotide mimetics. U.S. Pat. No. 5,652,355, HybridOligonucleotide Phosphorothioates, issued Jul. 29, 1997, and U.S. Pat.No. 5,652,356, Inverted chimeric and Hybrid Oligonucleotides, issuedJul. 29, 1997, which describe the synthesis and effect ofphysiologically-stable antisense molecules, are incorporated byreference. O/E-1 gene antisense molecules may be introduced into cellsby microinjection, liposome encapsulation or by expression from vectorsharbouring the antisense sequence.

DESCRIPTION OF THE DRAWINGS

The invention will now be illustrated by the following non-limitingExamples in which:

FIG. 1. O/E-1 is expressed already in undifferentiated 3T3-L1preadipocytes. RT-PCR analysis of O/E-1 levels in mouse 3T3-L1preadipocytes differentiated for 0, 1, 2, 4, 6 and 8 days usingdexamethasone, insulin and isobutylmetylxanthine as inducers, the datashown is after 30 cycles of PCR (lower panel). Mouse 36B4 PCR primerswere used for control of cDNA levels, the data shown is after 20 cyclesof PCR (upper panel). RNA from mouse 70Z/3 pre-B-cells and liver wereused as positive and negative controls for O/E-1 expression. +RTindicates that the cDNA synthesis was performed in the presence ofreverse transcriptase while −RT indicates that the cDNA synthesis wasperformed in the absence of reverse transcriptase.

FIG. 2. O/E-1 is expressed in human adipose tissue. RT-PCR analysis ofO/E-1 levels in human adipose tissue, data shown is after 35 cycles ofPCR (lower panel). 36B4 primers were used for control of cDNA levels,data shown is after 25 cycles of PCR (upper panel). RNA from human Nalm6 pre-B-cells and Jurkat T-cells were used as positive and negativecontrols for O/E-1 expression. +RT indicates that the cDNA synthesis wasperformed in the presence of reverse transcriptase while −RT indicatesthat the cDNA synthesis was performed in the absence of reversetranscriptase.

FIG. 3. O/E-1 promotes adipocyte differentiation in uncommitted NIH/3T3fibroblasts. Differentiation of vector, O/E-1, and PPARγ2 retrovirusinfected NIH/3T3 mouse fibroblasts, induced to differentiate usingdexamethasone, insulin and isobutylmethylxanthine either in the presenceor absence of the PPARγ ligand darglitazone and were stained with OilRed O after 10 days of differentiation.

FIG. 4. O/E-1 increases the rate of differentiation in 3T3-L1preadipocytes. Differentiation of vector and O/E-1 infected 3T3-L1preadipocytes. A. Infected cells induced with dexamethasone only stainedwith Oil Red O after indicated days of differentiation. B. Infectedcells induced with dexamethasone and the PPARγ ligand darglitazone OilRed O stained after indicated days of differentiation.

FIG. 5. O/E-1 enhances the rate of differentiation in untransformedmouse embryonic fibroblasts (MEF). Cells were infected with vector orO/E-1 retrovirus and induced to differentiate with dexamethasone,insulin, isobutylmethylxanthine and the PPARγ ligand darglitazone andstained with Oil Red O after 10 days.

FIG. 6. The ability of human O/E-1 to stimulate adipocytedifferentiation was tested in MEF. Duplicate dishes with cells infectedwith vector or human O/E-1 were induced to differentiate as described inexample 3, with or without darglitazone present.

FIG. 7. Expression levels of O/E-1, O/E-2 and O/E-3 transcriptionfactors and markers of adipate differentiation in adipose tissue, liver,B-lymphoid cell lines (A) and differentiated 3T3-L1 preadipocytes (B).

FIG. 8. mRNA levels of markers for adipocyte differentiation in vectorand O/E-1 infected cultures. Total RNA was extracted from cultures atpreconfluence (p.c), confluence (c), day 1, 2 and 4 (d1-d4) postinduction. After Dnase treatment and cDNA synthesis mRNA levels wasdetermined by real-time PCR. 36B4 mRNA was used to calculate 2^(−□Ct)values.

FIG. 9. O/E-1, -2 and -3 gene expression is regulated in lean and obesemice.

FIG. 10. A dominant negative form of O/E-1 reduces adipogenesis invitro. 3T3-L1 preadipocytes were infected with a retrovirus vector aloneor retroviruses harboring a dominant negative form of O/E-1 where atruncated cDNA, encoding only the DNA-binding domain of O/E-1, was fusedto the repressor domain of engrailed. After puromycin selection cellswere induced to adipocyte differentiation by dexamethasone, insulin and3-isobutyl-1-methylxanthine two days post confluence. Adipogeneis wasassessed by Oil Red O staining 5 days post induction. Petri dishes (60mm) and micrographs are shown.

EXAMPLE 1

Expression of O/E-1 during Adipate Differentiation

3T3-L1 preadipocytes (ATCC) were grown to confluence and allowed todifferentiate to adipocytes two days post confluence using theadipogenic inducers dexamethasone (1 μM) insulin (1 μg/ml) andisobutylmethylxanthine (0.115 mg/ml) (DIM) for two days and insulin onlyfor two days. RNA was prepared from confluent non-induced cells, andfrom induced cells at day 1, 2, 4, 6 and 8 post induction. cDNA wasprepared and the amount of O/E-1 and 36B4 control transcript wasdetermined by PCR (FIG. 1). Interestingly, O/E-1 was expressed alreadyin uninduced 3T3-L1 cells, indicating that this factor is present earlyin the adipocyte differentiation program before the onset of otheradipogenic factors such as PPARγ and C/EBP (Cowherd et al., 1999). Aslight upregulation (3-4 fold) in the O/E-1 transcript levels could alsoobserved during adipocyte differentiation, while no expression could bedetected in the liver. (FIG. 1.) These results indicate that O/E-1 isexpressed at early stages in adipocyte differentiation and may playimportant roles for the development of this cell lineage.

EXAMPLE 2

Expression of O/E-1 in Human Adipate Tissue

All functions of O/E-1 in B-lymphocyte studied so far seem to beconserved between mice and humans, suggesting that the same situation istrue also for O/E-1 in adipose tissue. To determine if O/E-1 isexpressed in human adipose tissue total RNA was prepared and used astemplate for cDNA synthesis. As control for O/E-1 expression, cDNAs fromhuman B and T-lymphocytes were also included. O/E-1 and 36B4 controltranscript levels were determined by PCR (FIG. 2) and O/E-1 was indeedexpressed also in human adipose tissue at levels comparable to thosefound in human B-lymphocytes, (FIG. 2.) indicating that the role O/E-1may play in mouse adipose tissue is persevered also in humans.

EXAMPLE 3

Overexpression of O/E-1 by Retroviral Transfer

A classical way of studying the adipocyte differentiating potential of agene is to overexpress it in either uncommitted NIH/3T3 fibroblasts orin 3T3-L1 preadipocytes using retroviral transfer. The adipocytedifferentiating potential can then be assayed by microscopic examinationand by staining with lipophilic dyes such as Oil Red O. O/E-1 was thusexpressed in mouse NIH/3T3 fibroblasts, 3T3-L1 preadipocytes and mouseembryonic fibroblasts to investigate its adipocyte differentiatingpotential. cDNAs encoding mouse O/E-1, human O/E-1 and mouse PPARγ2 werecloned in the pBabepuro retrovirus vector. These retrovirus vectors aswell as the empty vector were transiently transfected into Phoenix highefficiency packaging cells. Retroviruses containing supernatants wereused to infect NIH/3T3 mouse fibroblasts. After puromycin (2 μg/ml)selection for two days, cells were allowed to grow to confluence afterwhich adipogenic inducers were added (DIM, see above), after 10 dayscells were stained with the lipophilic dye Oil Red O. As expected PPARγinduced some differentiation of NIH/3T3 mouse fibroblasts intolipid-containing cells that resembled cultured adipocytes in thepresence of adipogenic inducers (FIG. 3). Also as expected a dramaticincrease was observed when the high affinity PPARγ ligand darglitazone(0.5 μM) was present throughout the experiment in combination with theadipogenic inducers. No differentiation was observed in NIH/3T3 vectorinfected cells treated with adipogenic inducers either in the presenceor absence of darglitazone. Surprisingly NIH/3T3 cells infected withmouse O/E-1 development to adipocytes at a similar rate as PPARγ2infected cells and this process was also greatly enhanced whendarglitazone was present, suggesting that O/E-1 is involved incontrolling adipocyte differentiation (FIG. 3). The ability of humanO/E-1 to induce adipocyte differentiation of NIH/3T3 was also tested andfound to be similar as that of mouse O/E-1 (Data not shown).

The fibroblastic 3T3-L1 preadipocyte cells have the capability todifferentiate to adipocytes when given the proper hormonal stimulation,usually a combination of dexamethasone, insulin andisobutylmethylxanthine, as opposed to NIH/3T3 where overexpression ofadipocyte differentiating factors is necessary to get adipocytedevelopment. Untransformed mouse embryonic fibroblasts (MEF) can also beforced to adipocyte differentiation using the same adipogenic factors;the efficiency is however much lower than with 3T3-L1. To show that thecapability of O/E-1 to stimulate adipocyte differentiation was notspecific for one particular cell line, O/E-1 was over expressed usingretrovirus mediated transfer in 3T3-L1 and MEF. Stable clones wereselected 2-3 days using puromycin (2 μg/ml) and surviving cells weregrown to confluence. 3T3-L1 cells differentiate to adipocytes at highrate when all adipogenic inducers are present and this could make itdifficult discriminate the effect of an additional adipogenic signal.The cells were therefore induced with dexamethasone (1 μM) alone for twodays or in combination with darglitazone (0.5 μM) throughout theexperiment to get a low differentiation rate. MEF on the other handdevelop into adipocytes quite inefficiently and were thus induced withthe more powerful adipogenic combination of dexamethasone (1 μM),insulin (5 μg/ml) isobutylmethylxanthine (0.115 mg/ml) and darglitazone(0.5 μM) for two days, insulin and darglitazone for two days anddarglitazone alone all through the experiment. O/E-1, or vector infected3T3-L1 preadipocytes were stained with Oil Red O at day 9 and 10 postinduction (FIG. 4A) and the rate of adipocyte differentiation wasclearly enhanced by O/E-1 under these very slow differentiationconditions. When darglitazone was added in combination withdexamethasone the overall rate of differentiation was enhanced and thecells were stained at day 3, 4 and 7. At day 4 and 7 the O/E-1 infectedcultures contained far more adipocytes than vector infected cultures(FIG. 4B). The same pattern could also be observed in MEFs where somedifferentiation into adipocytes could be observed 10 days post inductionin vector infected cultures, the adipocyte like cells grew in many smallclusters of 20-50 cells (FIG. 5). A dramatic difference was observed inO/E-1 infected cultures where many cells in large clusters showed OilRed O staining.

EXAMPLE 4

Assays for Measuring O/E-1 Activity

Mutagenesis of the DNA binding motif of O/E-1 indicate that severalhistidines and cysteins are important for DNA binding and that theseamino acids could represent a loop that is stabilized by a centraldivalent metal cation (Hagman et al., 1995). Renaturation of denaturedO/E-1 polypeptides in the presence of various divalent cations did alsoshow that DNA binding to a ³²P labeled probe in electrophoretic mobilityshift assay (EMSA) was dependent upon inclusion of Zn²⁺ ions. DNAbinding could also be achieved in the presence of Cd²⁺ and to a lesserextent by inclusion of Mg²⁺ ions (Hagman et al., 1995) suggesting thatother ions could displace Zn²⁺ in vitro. The O/E-1 amino acid sequencecan not be aligned with consensus metal binding motifs like zinc fingersindicating that O/E-1 harbors a unique metal containing DNA bindingmotif that mediates DNA interaction. This unique domain of O/E-1 couldbe a possible target for modulation of the DNA binding capability of theprotein. Relative high concentration Zn²⁺ is needed in vitro to achieveoptimal O/E-1 DNA binding (1-10 μM) suggesting that this may be alimiting factor for DNA binding in vivo. Development of high affinitylow molecular weight compounds could thus be an attractive way ofmodulating the O/E-1 activity and adipocyte differentiation.Recombinant, truncated O/E-1 protein containing the DNA binding anddimerization domains can be used in EMSA. Using a protein binding bufferdevoid of Zn² ⁺ions, which gives very poor binding to natural O/E-1 DNAbinding sites, can be used for testing low molecular weight compoundsfor modulation, especially stimulation of DNA binding capacity of O/E-1in EMSA. Using a protein binding buffer with physiological, or close tophysiological concentration of Zn²⁺ ions, which gives effective bindingto natural O/E-1 DNA binding sites, can be used for testing lowmolecular weight compounds for modulation, especially inhibition of DNAbinding capacity of O/E-1 in EMSA. To increase the throughput otherassays based on fluorescence energy transfer for measuring O/E-1 bindingto its DNA target site can be developed. Compounds isolated based ontheir capacity to modulate O/E-1 DNA binding can be tested further incell based assays using reporter constructs containing natural DNAbinding sites. Selected compounds with activities in binding andreporter assays can be tested for activity in adipocyte differentiationmodels based on preadipocyte cell lines.

EXAMPLE 5

Overexpression of Human O/E-1 in MEF

The ability of human O/E-1 to stimulate adipocyte differentiation wastested in MEF. Duplicate dishes with cells infected with vector or humanO/E-1 were induced to differentiate as described in example 3, with orwithout darglitazone present (FIG. 6). At day 13 post-induction adipatedifferentiation was measured by Oil Red O staining. Vector infectedcells differentiated to adipocytes with low efficiency when treated withvehicle. As expected, the addition of darglitazone increased the numberof cells that developed into adipocytes. Cultures infected with humanO/E-1 did, analogues to mouse O/E-1, show increased number of adipocytescompared to vector infected cultures even in the absence ofdarglitazone. As observed with mouse O/E-1, cultures infected with humanO/E-1 showed even further enhanced differentiation when darglitazone waspresent. These results indicate that mouse and human O/E-1 have asimilar capacity to promote adipocyte differentiation.

EXAMPLE 6

Expression of O/E-1, O/E-2 and O/E-3 in Adipose Tissue, Liver,B-Lymphoid Cell Lines and Differentiated 3T3-L1 Preadipocytes

Total RNA was isolated from mouse visceral adipose tissue, liver and the70Z/3 B-lymphoid cell line using RNA STAT-60 (BioSite) according to themanufacturer's instructions. DNA was removed from RNA preparations(DNA-free Kit, Ambion) and first-strand synthesis was performed usingrandom primers (Superscript First-Strand Synthesis System for RT-PCR,Gibco BRL). Transcript levels of O/E-1, -2, and 3 were analyzed byreal-time PCR using a ready-to-use assay based on the Taqman technology(PE Applied Biosystems, Weiterstadt, Germany). Fluorescent SYBER-Greenwas used for detection. 36B4 mRNA was used to calculate 2^(−ΔCt) values.

Real-time PCR analysis using primers for O/E-1, -2, and -3 on cDNAsderived from mouse adipose tissue, liver and the 70Z/3 B-lymphoid cellline revealed that all three isoforms of O/E are expressed in mouseadipose tissue, although at different levels with O/E-1 being mostabundant (FIG. 7A). Consistent with work reported by others, O/E-1 wasthe only O/E gene expressed in B-lymphoid cells, while the liver wasnegative for all isoforms.

3T3-L1 preadipocytes were cultured and induced to differentiate toadipocytes using dexamethasone, insulin, and isobutylmethylxanthine atday 0. RNA was extracted from cultures at preconfluence (p.c),confluence (c), and day 1, 2, 4, 6 and 8 (d1-d8) post induction. DNasetreated RNA samples were used as templates in cDNA synthesis, which wereused to determine expression levels of O/E-1, -2,-3, PPARγ1 and 2,SREBP1, C/EBPα, aP2 and GPDH by real-time PCR. 36B4 mRNA was used tocalculate 2^(−ΔCt) values.

Next, the 3T3-L1 model system for adipocyte differentiation was used todetermine at which stage during adipocyte development O/E geneexpression of was initiated, and if expression was regulated during thecoarse of differentiation. 3T3-L1 preadipocytes were grown to confluenceand allowed to differentiate to adipocytes two days post confluenceusing the adipogenic inducers dexamethasone (1 μM) insulin (1 μg/ml) andisobutylmethylxanthine (0.5 mM) (DIM) for two days and insulin only fortwo additional days. mRNA levels for O/E-1, 2 and 3, as well as forknown markers of adipocyte differentiation, were determined by real-timePCR (FIG. 7B). All O/E-forms were expressed already in uninducedpreconfluent 3T3-L1 cells, indicating that these factors are present atearly stages of adipocyte development. O/E-2 and 3 were however presentat lower levels than O/E-1, which is coherent with the pattern observedin mouse adipose tissue. During 3T3-L1 differentiation O/E-1 wasupregulated approximately 8 fold and this tendency could also beobserved with the other isoforms although the upregulation was only 2-3fold. To compare the expression pattern of the O/Es with known markersof adipocyte development we measured mRNA levels of PPARγ1, PPARγ2,SREBP1, C/EBPα, aP2 and GPDH by real-time PCR (FIG. 7B). This analysisshowed that the O/Es were expressed at an early stage when low levels ofSREBP and PPARγ1 are detected but before the onset of C/EBPα and PPARγ2.These results indicates that O/E-1 mRNA levels are modulated duringadipogenesis with a profile similar to those of PPARγ1 and SREBP1.

Real-time PCR analysis oligonucleotides Forward oligo Reverse oligo SEQID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO:6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 11 SEQID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ IDNO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20

EXAMPLE 7

mRNA Levels of Markers for Adipocye Differentiation in Vector and O/E-1Infected Cells

To study the mechanism behind the adipogenic potential of O/E-1,real-time PCR was performed on cDNAs derived from 3T3-L1 cells infectedwith vector or O/E-1 retroviruses collected at various time-pointsduring dexamethasone (1 μM) and darglitazone (0.5 μM) stimulateddifferentiation (FIG. 8). At early stages during differentiation, weobserved no significant changes in mRNA levels of any of the studiedadipocyte differentiation marker genes. By day four-post induction therewas, however, a clear up regulation of most of the marker genes in O/E-1infected cultures, implying that O/E-1 is not directly controlling anyof the examined genes. The upregulation of these genes at later stagesis likely due to a more efficient differentiation of O/E-1 infectedcells.

EXAMPLE 8

O/E-1, -2 and -3 Gene Expression is Regulated in Lean and Obese Mice

O/E-1, -2 and -3 gene expression is regulated in lean and obese mice.9-10 wk C57/BL6 mice were fed a standard laboratory rodent chow or chowplus unlimited access to cafeteria diet for 11 wk. High gainers wereseparated from low gainers by the weight they gained. Five high gainerswere compared with five mice that were fed a standard diet. Mousevisceral adipose tissue was isolated and total RNA was extracted andtreated with DNase. O/E-1, -2 and -3 mRNA levels were determined byreal-time RT-PCR 36B4 mRNA was used to calculate 2^(−ΔCt) values (FIG.9).

EXAMPLE 9

A Dominant Negative Form of O/E-1 Reduces Adipogenesis In Vitro

If the O/E proteins are important for adipocyte differentiation itshould be possible to reduce differentiation of 3T3-L1 by overexpression of a dominant negative form of O/E-1. This would also affectthe function of all three O/E proteins since they are reported tointeract with the same consensus DNA-binding site (A. Travis, J. Hagman,L. Hwang, R. Grosschedl, Mol. Cell. Biol. 13, 3392-3400 (1993); J.Hagman, M. J. Gutch, H. Lin, R. Grosschedl, Embo J 14, 2907-16 (1995).Constructing a dominant negative form of the O/E-1 protein iscomplicated by the fact that this transcription factor has a secondtransactivating domain within the DNA-binding domain. To overcome thisproblem we fused the repressor domain of engrailed (amino acids 1-298)with a truncated O/E-1 cDNA lacking the C-terminal transactivationdomain while retaining the DNA-binding and dimerization domains (M.Sigvardsson, Mol Cell Biol 20, 3640-54. (2000). Retroviruses carryingO/E-1-engrailed were used to infect 3T3-L1 cells and the differentiationcapability was compared with vector infected cells. Differentiation wasinduced with Dexamethasone (1 μM), 3-isobutyl-1-methylxanthine (0.5 mM)and Insulin (1 μg/ml) and cultures were stained with Oil Red O after 5days. As shown in FIG. 7, over expression of the dominant negative formof O/E-1 in 3T3-L1 cells reduced the capacity of these cells to developinto adipocytes. There was not an absolute block of differentiation inO/E-1-engrailed infected cultures because some cells still accumulatedlipids but the number of differentiating cells was reduced. These dataindicate that O/E target genes are important and required fordifferentiation of 3T3-L1 preadipocytes into adipocytes.

1. A method for identifying a compound which modulates pre-adipocytedifferentiation comprising testing whether the compound modulates theactivity and/or amount of O/E-1 and/or O/E-2 and/or O/E-3.
 2. A methodaccording to claim 1, comprising testing whether the compound modulatesthe activity and/or amount of O/E-1.
 3. A method according to claim 1,comprising testing whether the compound modulates the activity and/oramount of O/E-2.
 4. A method according to claim 1, comprising testingwhether the compound modulates the activity and/or amount of O/E-3.
 5. Amethod according claim 2, in which the test is independently selectedfrom: i) measurement of O/E-1 activity using a reporter gene assaycomprising a cell line which expresses O/E-1 and a reporter gene coupledto an O/E-1 response element and assaying for expression of the reportergene; ii) measurement of O/E-1 activity using purified O/E-1 protein ora fragment thereof, and assaying the interaction between O/E-1 and a DNAfragment; iii) measurement of O/E-1 activity using purified O/E-1protein or a fragment thereof and a dimerisation partner or a fragmentthereof, and assaying the dimerisation of O/E-1; and iv) measurement ofO/E-1 transcription or translation in a cell line expressing O/E-1, v)measurement of direct compound binding or competitive binding to O/E-1.6. A method according to claim 5, in which measurement of O/E-1 activityusing purified O/E-1 protein or a fragment thereof, and assaying theinteraction between O/E-1 and a DNA fragment uses an electrophoresismobility shift assay (EMSA).
 7. A method according to claim 5, in whichmeasurement of O/E-1 activity using purified O/E-1 protein or a fragmentthereof and a dimerisation partner or a fragment thereof, and assayingthe dimerisation of O/E-1 using time resolved fluorescence resonanceenergy transfer or scintillation proximity assay.
 8. A method accordingto claim 5, in which the cell line is independently selected from a3T3-L1 preadipocyte cell, a 3T3-L1 adipocytes cell, a NIH/3T3fibroblast, or an embryonic fibroblast.
 9. A method according to claim5, in which the O/E-1 protein is human recombinant O/E-1 or mouserecombinant O/E-1.
 10. A method according to claim 3, in which the testis independently selected from: i) measurement of O/E-2 activity using areporter gene assay comprising a cell line which expresses O/E-2 and areporter gene coupled to an O/E-2 response element and assaying forexpression of the reporter gene; and ii) measurement of O/E-2transcription or translation in a cell line expressing O/E-2.
 11. Amethod according to claim 4, in which the test is independently selectedfrom: i) measurement of O/E-3 activity using a reporter gene assaycomprising a cell line which expresses O/E-3 and a reporter gene coupledto an O/E-3 response element and assaying for expression of the reportergene; and ii) measurement of O/E-3 transcription or translation in acell line expressing O/E-3.
 12. A method according to claim 10 or 11, inwhich the cell line is independently selected from a 3T3-L1 preadipocytecell or a 3T3-L1 adipocytes cell.
 13. A method according claim 10 or 11,in which the O/E-2 protein is mouse recombinant O/E-2 and the O/E-3protein is mouse recombinant O/E-3.
 14. A method according to claim 5,in which direct compound binding or competitive binding to O/E-1 ismeasured by time resolved fluorescence resonance energy transfer orscintillation proximity assay.